In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as C. elegans, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Caspases, a class of proteins central to the apoptotic program, are cysteine protease having specificity for aspartate at the substrate cleavage site. These proteases are primarily responsible for the degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. For example, one of the caspases identified in humans was previously known as the interleukin-1xcex2 (IL-1xcex2) converting enzyme (ICE), a cysteine protease responsible for the processing of pro-IL-1xcex2 to the active cytokine. Overexpression of ICE in Rat-1 fibroblasts induces apoptosis (Miura et al., Cell 75:653 [1993]).
Many caspases and proteins that interact with caspases possess domains of about 60 amino acids called a caspase recruitment domain (CARD). Hofmann et al. (TIBS 22:155 [1997]) and others have postulated that certain apoptotic proteins bind to each other via their CARDs and that different subtypes of CARDs may confer binding specificity, regulating the activity of various caspases, for example.
The functional significance of CARDs have been demonstrated in two recent publications. Duan et al. (Nature 385:86 [1997]) showed that deleting the CARD at the N-terminus of RAIDD, a newly identified protein involved in apoptosis, abolished the ability of RAIDD to bind to caspases. In addition, Li et al. (Cell 91:479 [1997]) showed that the N-terminal 97 amino acids of apoptotic protease activating factor-1 (Apaf-1) was sufficient to confer caspase-9-binding ability.
The invention relates to the discovery of two genes, card-3 (cDNA in FIG. 2, SEQ ID NO:2) and card-4 (partial cDNA in FIG. 4, SEQ ID NO:4), which encode the CARD-containing proteins CARD-3 (FIG. 1, SEQ ID NO:1) and CARD-4 (partial sequence in FIG. 3, SEQ ID NO:3).
CARD-3 contains a kinase domain at amino acid positions 1-300, followed by a linker domain at positions 301-431 and a CARD at positions 432-540. In the CARD-4 partial amino acid sequence (FIG. 3, SEQ ID NO:3), a CARD is located at positions 3-72, counting the first Ser in the sequence as the first position.
Like other proteins containing a CARD, CARD-3 and CARD-4 are expected to participate in the network of interactions that lead to caspase activation. Accordingly, the human card-3 and card-4 genes described herein may be useful in diagnostic and therapeutic applications directed to the regulation of cell growth and death such as the diagnosis of certain cancers, e.g., breast cancer and colorectal cancer. These genes and the proteins they encode can be used to screen for molecules which inhibit or enhance CARD-3 or CARD-4 activity. Moreover, agonist ligands which bind to the proteins encoded by these genes may be useful for the treatment of certain cancers.
The invention encompasses methods of diagnosing and treating patients who are suffering from a disorder associated with an abnormal level or rate (undesirably high or undesirably low) of apoptotic cell death, abnormal activity of the Fas/APO-1 receptor complex, abnormal activity of the TNF receptor complex, or abnormal activity of a caspase by administering a compound that modulates the expression of CARD-3 and/or CARD-4 (at the DNA, mRNA or protein level, e.g., by altering mRNA splicing) or by altering the activity of CARD-3 and/or CARD-4. Examples of such compounds include small molecules, antisense nucleic acid molecules, ribozymes, and polypeptides.
Certain disorders are associated with an increased number of surviving cells, which are produced and continue to survive or proliferate when apoptosis is inhibited. These disorders include cancer (particularly follicular lymphomas, carcinomas associated with mutations in p53, and hormone-dependent tumors such as breast cancer, prostate cancer, and ovarian cancer), autoimmune disorders (such as systemic lupus erythematosis, immune-mediated glomerulonephritis), and viral infections (such as those caused by herpesviruses, poxviruses, and adenoviruses).
Failure to remove autoimmune cells that arise during development or that develop as a result of somatic mutation during an immune response can result in autoimmune disease. One of the molecules that plays a critical role in regulating cell death in lymphocytes is the cell surface receptor for Fas.
Populations of cells are often depleted in the event of viral infection, with perhaps the most dramatic example being the cell depletion caused by the human immunodeficiency virus (HIV). Surprisingly, most T cells that die during HIV infections do not appear to be infected with HIV. Although a number of explanations have been proposed, recent evidence suggests that stimulation of the CD4 receptor results in the enhanced susceptibility of uninfected T cells to undergo apoptosis.
A wide variety of neurological diseases are characterized by the gradual loss of specific sets of neurons. Such disorders include Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and various forms of cerebellar degeneration. The cell loss in these diseases does not induce an inflammatory response, and apoptosis appears to be the mechanism of cell death.
In addition, a number of hematologic diseases are associated with a decreased production of blood cells. These disorders include anemia associated with chronic disease, aplastic anemia, chronic neutropenia, and the myelodysplastic syndromes. Disorders of blood cell production, such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow. These disorders could result from the activation of genes that promote apoptosis, acquired deficiencies in stromal cells or hematopoietic survival factors, or the direct effects of toxins and mediators of immune responses.
Two common disorders associated with cell death are myocardial infarctions and stroke. In both disorders, cells within the central area of ischemia, which is produced in the event of acute loss of blood flow, appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted time period and morphologically appear to die by apoptosis.
The invention features isolated nucleic acid molecules (i.e., a nucleic acid molecule that is separated from the 5xe2x80x2 and 3xe2x80x2 coding sequences of other genes with which it is immediately contiguous in the naturally occurring genome of an organism, also referred to as a recombinant nucleic acid molecule) that encodes a CARD-3 or CARD-4 polypeptide. Within the invention are polypeptides having the sequence of SEQ ID NO:1 or SEQ ID NO:3, or encoded by nucleic acid molecules having the sequence shown in SEQ ID NO:2 or SEQ ID NO:4. However, the invention is not limited to nucleic acid molecules and polypeptides that are identical to those SEQ ID Nos 1-4. For example, the invention includes nucleic acid molecules which encode splice variants, allelic variants, mutant forms, and full-length forms of card-3 or card-4 as well as the proteins encoded by such nucleic acid molecules.
Also within the invention are nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO:2 or SEQ ID NO:4. Such molecules include, for example, other mammalian homologues of card-3 or card-4. As described further below, molecules that are substantially identical to those of SEQ ID Nos 1-4 are also encompassed by the invention.
Accordingly, the invention relates to isolated nucleic acid molecules which encode CARD-3 or CARD-4 polypeptides or variants thereof. Such polypeptides or fragments thereof can include the amino acid sequence of SEQ ID NO:1, the amino acid sequence of SEQ ID NO:3, at least 15 contiguous amino acids of SEQ ID NO:1, at least 15 contiguous amino acids of SEQ ID NO:3, amino acids from about 1 to about 300 (e.g., amino acids 20 to 320) of SEQ ID NO:1 (kinase domain); amino acids from about 301 to about 341 (e.g., amino acids 281 to 361 or 321 to 341) of SEQ ID No:1 (linker domain); amino acids from about 432 to about 540 (e.g., amino acids 412 to 560 or 452 to 520) of SEQ ID NO:1 (CARD domain), or amino acids from about 3 to about 72 (e.g., amino acids 1 to 92 or 23 to 52) of SEQ ID NO:3 (CARD domain).
In addition, the isolated nucleic acid molecules of the invention can include the nucleotide sequence of SEQ ID NO:2 or 4 (or the RNA variants thereof), a nucleotide sequence complementary to SEQ ID NO:2 or 4, or a nucleic acid fragment of any previous nucleic acid molecule of at least 15 nucleotides in length. The isolated nucleic acid molecules further include nucleic acid molecules which encode polypeptides that are at least 80% identical to SEQ ID NO:1, nucleic acid molecules which encode polypeptides that are at least 80% identical to SEQ ID NO:3, nucleic acid molecules which hybridize under stringent conditions to the nucleic acid molecule of SEQ ID NO:2, nucleic acid molecules which hybridize under stringent conditions to the nucleic acid molecule of SEQ ID NO:4, nucleic acid molecules which hybridize under stringent conditions to the cDNA sequence contained within ATCC Accession No. 203037.
The invention further relates to methods for detecting the presence of any nucleic acid molecule described above by contacting a sample (e.g., one containing mRNA molecules) suspected of containing the nucleic acid molecule with a nucleic acid probe which selectively hybridizes to the nucleic acid molecule and determining whether the nucleic acid probe binds to the nucleic acid molecule in the sample. A nucleic acid probe is a nucleic acid molecule or analog thereof which is capable of sequence specific binding to a target nucleic acid. Such probes can be labeled with a detectable label (e.g., a chromogenic converting enzyme such as horseradish peroxidase or a radioisotope).
The invention also features substantially pure CARD-3 and CARD-4 polypeptides and fragments thereof, each polypeptide or fragment having at least one functional domain of CARD-3 or CARD-4 (e.g., the kinase domain, linker domain, or CARD of CARD-3; or the CARD of CARD-4).
Thus, the invention relates to substantially pure polypeptides having the amino acid sequence of SEQ ID NO:1, having the amino acid sequence of SEQ ID NO:3, having at least 15 contiguous amino acids of SEQ ID NO:1, having at least 15 contiguous amino acids of SEQ ID NO:3, having amino acids from about 1 to about 300 of SEQ ID NO:1 (kinase, domain), having amino acids from about 301 to about 431 of SEQ ID NO:1 (linker domain); having amino acids from about 432 to about 540 of SEQ ID NO:1 (card domain), or having amino acids from about 3 to about 72 of SEQ ID NO:3 (card domain). In addition, the invention features methods for producing the aforementioned substantially pure polypeptides of the invention by culturing a host cell (e.g., a bacterium) containing an aforementioned nucleic acid molecule which expresses the polypeptide
This invention also features hybrid molecules comprising a polypeptide of the invention fused to a heterologous polypeptide (e.g., the CARD of CARD-3 fused to xcex2-galactosidase).
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given compound (e.g., a CARD-3 or CARD-4 polypeptide) means that the compound is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the compound may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). When chemically synthesized, a compound of the invention is substantially pure when it is substantially free from the chemical compounds used in the process of its synthesis. Polypeptides or other compounds of interest are substantially free from other compounds when they are within preparations that are at least 60% by dry weight the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides (e.g., 150, 200, 250, or 300 nucleotides).
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 with the default parameters as specified therein.
The BLAST programs, provided as a service by the National Center for Biotechnology Information are very useful for making sequence comparisons. The programs are described in detail by Karlin et al. (Proc Natl Acad Sci USA 87:2264 [1990] and Proc Natl Acad Sci USA 90:5873 [1993]) and Altschul et al. (Nucl Acids Res 25:3389 [1997]) and are available on the Internet at the URL: http://www.ncbi.nlm.nih.gov.
The invention also features a host cell that includes an isolated nucleic acid molecule encoding CARD-3 or CARD-4 (either alone or in conjunction with a heterologous polypeptide, such as a detectable marker), or a nucleic acid vector that contains a sequence encoding CARD-3 or CARD-4 (again, with or without a heterologous polypeptide). The vector can be an expression vector, and can include a regulatory element.
An antibody that specifically binds a CARD-3 or CARD-4 polypeptide is also within the scope of the present invention and is useful, for example, to detect CARD-3 or CARD-4 in a biological sample, or to alter the activity of CARD-3 or CARD-4. For example, CARD-3 or CARD-4 can be detected in a biological sample by contacting the sample with an antibody that specifically binds CARD-3 or CARD-4 under conditions that allow the formation of a CARD-3 or CARD-4-antibody complex and detecting the complex, if present, as an indication of the presence of CARD-3 or CARD-4 in the sample. The use of-an antibody in a treatment regime, where it can alter the activity of CARD-3 or CARD-4, is discussed further below.
Accordingly, the invention features methods for detecting the presence of an aforementioned polypeptide by contacting the sample suspected of containing the polypeptide with a compound (e.g., an antibody) which selectively binds to the polypeptide and determining whether the compound binds to the polypeptide in the sample.
An antibody of the invention can be a monoclonal, polyclonal, or engineered antibody that specifically binds CARD-3 or CARD-4 (as described more fully below). An antibody that xe2x80x9cspecifically bindsxe2x80x9d to a particular antigen, for example, a CARD-3 or CARD-4 polypeptide of the invention, will not substantially recognize or bind to other molecules in a sample, such as a biological sample, that includes CARD-3 or CARD-4. Thus, the invention also features methods for identifying a test compound (e.g., an antibody) which binds to a polypeptide of the invention by contacting the polypeptide with a test compound and determining whether the polypeptide binds to the test compound (e.g. by direct detection of the binding, detection of a competitor molecule which disrupts binding of the test compound to the polypeptide, and/or detection of binding using an assay for apoptosis activity).
Given that an object of the present invention is to alter the expression or activity of card-3 or card-4 in vivo, a pharmaceutical composition containing, for example, isolated nucleic acid molecules encoding CARD-3 or. CARD-4 proteins (or fragments thereof), nucleic acid molecules that are antisense to card-3 or card-4 (i.e., that have a sequence that is the reverse and complement of a portion of the coding strands of card-3 or card-4 genes), CARD-3 or CARD-4 polypeptides, or antibodies, small molecules, or other compounds that specifically bind CARD-3 or CARD-4 polypeptides are also a feature of the invention.
The discovery and characterization of card-3 and card-4 and the polypeptides they encode makes it possible to determine whether a given disorder is associated with aberrant expression of card-3 or card-4 (meaning expression at the level of gene transcription or mRNA translation) or activity of CARD-3 or CARD-4. For example, one can diagnose a patient as having a disorder associated with aberrant expression of card-3 or card-4 by measuring card-3 or card-4 expression in a biological sample obtained from the patient. An increase or decrease in card-3 or card-4 expression in the biological sample, compared with card-3 or card-4 expression in a control sample (e.g., a sample of the same tissue collected from one or more healthy individuals) indicates that the patient has a disorder associated with aberrant expression of card-3 or card-4. Similarly, one can diagnose a patient as having a disorder associated with aberrant activity of CARD-3 or CARD-4 by measuring CARD-3 or CARD-4 activity in a biological sample obtained from the patient. An increase or decrease in CARD-3 or CARD-4 activity in the biological sample, compared with CARD-3 or CARD-4 activity in a control sample, indicates that the patient has a disorder associated with aberrant activity of CARD-3 or CARD-4. The techniques required to measure gene expression or polypeptide activity are well known to those of ordinary skill in the art.
Thus, the invention features (1) methods for diagnosing a patient as having a disorder associated with aberrant expression of card-3 or card-4 by measuring card-3 or card-4 expression in a biological sample obtained from the patient where increased or decreased card-3 or card-4 expression in the biological sample compared with card-3 or card-4 expression in a control sample indicates that the patient has a disorder associated with aberrant expression of card-3 or card-4; (2) methods for diagnosing a patient as having a disorder associated with expression of an isoform of CARD-3 or CARD-4 by isolating card-3 or card-4 mRNA or CARD-3 or CARD-4 polypeptide from the patient and determining the sequence of the mRNA or polypeptide where a difference in the sequence as compared to the nucleotide sequence of SEQ ID NO:2 or 4 or the polypeptide sequence of SEQ ID NO:1 or 3 indicates expression of an isoform of CARD-3 or CARD-4; and (3) methods for diagnosing a patient as having a disorder associated with aberrant activity of CARD-3 or CARD-4 by measuring CARD-3 or CARD-4 activity in a biological sample obtained from the patient where increased or decreased CARD-3 or CARD-4 activity in the biological sample compared with CARD-3 or CARD-4 activity in a control sample indicates that the patient has a disorder associated with aberrant activity of CARD-3 or CARD-4.
In addition to diagnostic methods, such as those described above, the present invention encompasses methods and compositions for typing, evaluating the prognosis of, and appropriate treatment for, disorders associated with inappropriate expression of card-3 or card-4 or inappropriate activity of CARD-3 or CARD-4. For example, the nucleic acid molecules of the invention can be used as probes to classify cells in terms of their level of card-3 or card-4 expression, or as primers for diagnostic PCR analysis in which mutations, allelic variations, and regulatory defects in the card-3 or card-4 gene can be detected. Similarly, those of ordinary skill in the art can use routine techniques to identify inappropriate activity of CARD-3 or CARD-4, which can be observed in a variety of forms. For example, inappropriate activity can take the form of strong binding to inappropriate caspases. Diagnostic kits for the practice of such methods are also provided.
The invention further encompasses transgenic animals that express card-3 or card-4 and recombinant xe2x80x9cknock-outxe2x80x9d animals that fail to express card-3 or card-4. These animals can serve as new and useful models of disorders in which card-3 or card-4 expression is abnormal.
The invention also features antagonists and agonists of CARD-3 or CARD-4 that can inhibit or enhance, respectively, one or more of the biological activities of CARD-3 or CARD-4. Suitable antagonists can include small molecules (i.e., molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500), antibodies that specifically bind and xe2x80x9cneutralizexe2x80x9d CARD-3 or CARD-4 (as described below), and nucleic acid molecules that interfere with transcription or translation of card-3 or card-4 (e.g., antisense nucleic acid molecules and ribozymes). Agonists of CARD-3 or CARD-4 also include small and large molecules, and antibodies other than neutralizing antibodies.
Thus, the invention features (1) methods for modulating (e.g., decreasing or increasing) an activity (e.g., the ability to recruit caspases) of an aforementioned polypeptide of the invention by contacting a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide; and (2) methods of identifying a compound that modulates the activity (e.g., decrease or increase) of an aforementioned polypeptide having a CARD of CARD-3 or a CARD of CARD-4 by contacting the polypeptide with a test compound (e.g., polypeptides, ribonucleic acids, small molecules, ribozymes, antisense oligonucleotides, and deoxyribonucleic acids) and detecting and comparing the level of activity of the polypeptide in the presence or absence of the test compound.
The invention also features molecules that can increase or decrease the expression of card-3 or card-4 (e.g., by altering transcription or translation). Small molecules (as defined above), large molecules (as defined above), and nucleic acid molecules (e.g., antisense and ribozyme molecules) can be used to inhibit the expression of card-3 or card-4. Other types of nucleic acid molecules (e.g., molecules that bind to card-3 or card-4 negative transcriptional regulatory sequences) can be used to increase the expression of card-3 or card-4. Accordingly, the invention features methods for modulating apoptosis by modulating the expression or activity of a gene containing the sequence of an aforementioned nucleic acid of the invention encoding a CARD of CARD-3 or a CARD of CARD-4.
Compounds that modulate the expression of card-3 or card-4 in a cell can be identified by comparing the level of expression of card-3 or card-4 in the presence of a selected compound with the level of expression of card-3 or card-4 in the absence of that compound. A difference in the level of card-3 or card-4 expression indicating that the selected compound modulates the expression of card-3 or card-4 in the cell. A comparable test for compounds that modulate the activity of CARD-3 or CARD-4 can be carried out by comparing the level of CARD-3 or CARD-4 activity in the presence and absence of the compound. Accordingly, the invention features methods of identifying a compound that modulates the expression of a gene (e.g., a gene comprising the sequence of an aforementioned nucleic acid molecule of the invention) encoding a polypeptide having a CARD of CARD-3 or a CARD of CARD-4 by contacting the gene with a test compound (e.g., polypeptides, ribonucleic acids, small molecules, ribozymes, antisense oligonucleotide, and deoxyribonucleic acids) and detecting and comparing the level of expression of the gene in the presence and absence.
Patients who have a disorder mediated by abnormal CARD-3 or CARD-4 activity can be treated by administration of a compound that alters the expression of card-3 or card-4 or the activity of CARD-3 or CARD-4. When the objective is to decrease expression or activity, the compound administered can be a card-3 or card-4 antisense oligonucleotide or an anti-CARD-3 or anti-CARD-4 antibody, such as a neutralizing antibody, that specifically binds CARD-3 or CARD-4, respectively. Accordingly, the invention features methods for treating a patient having a disorder associated with the aberrant expression or activity any aforementioned polypeptide of the invention having a CARD of CARD-3 or a CARD of CARD-4 by administering a therapeutically effective amount of a compound (e.g., polypeptide, ribonucleic acid, small molecule, ribozyme, antisense oligonucleotide or deoxyribonucleic acid) that decreases or increases the expression or activity of the gene (e.g., a gene comprising the sequence of any nucleic acid molecule of the invention).
The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing or the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.